Diuretic and natriuretic polypeptides

ABSTRACT

This document provides diuretic and natriuretic polypeptides. For example, this document provides polypeptides having diuretic and/or natriuretic activities. In some cases, a polypeptide provided herein can have diuretic and natriuretic activities, while lacking the ability to lower blood pressure. This document also provides methods and materials for inducing diuretic and/or natriuretic activities within a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/376,880, filed on Apr. 15, 2010, which is a National Stageapplication under 35 U.S.C. §371 of International Application No.PCT/US2007/075465 having an International Filing Date of Aug. 8, 2007,which claims priority from U.S. Provisional Application No. 60/934,584,filed on Jun. 13, 2007, and U.S. Provisional Application Ser. No.60/836,581, filed on Aug. 8, 2006.

BACKGROUND

1. Technical Field

This document relates to methods and materials such as diuretic andnatriuretic polypeptides. For example, this document relates topolypeptides having diuretic and natriuretic activities while lackingthe ability to lower blood pressure.

2. Background Information

Members of the natriuretic polypeptide family are hormones that regulatebody fluid homeostasis. Atrial natriuretic peptide (ANP) is secreted byatrial myocytes in response to increased intravascular volume. Once ANPis in the circulation, its effects are primarily on the kidney, vasculartissue, and adrenal gland, in which its actions lead to the excretion ofsodium and water by the kidneys and a decrease in intravascular volumeand blood pressure. BNP also is of myocardial cell origin, and like ANP,it circulates in human plasma. BNP is natriuretic, rennin inhibiting,vasodilating, and lusitropic. The main circulating and storage form ofBNP is a 32 amino acid polypeptide with a ring structure. Physiologicalactions of BNP are mediated through a guanylate cyclase-linked receptor,natriuretic peptide receptor A (NPR-A). Clearance of BNP is promoted bya NPR-C receptor that removes it from the circulation. BNP also isdegraded through enzymatic cleavage by neutral endopeptidase. C-typenatriuretic peptide (CNP) is of endothelial cell origin and functions asa vasodilating and growth-inhibiting polypeptide. Dendroaspisnatriuretic peptide (DNP) is similar in structure to ANP, BNP, and CNP,and is isolated from the venom of Dendoaspis angusticeps or green mambasnake.

SUMMARY

This document relates to diuretic and natriuretic polypeptides. Forexample, this document provides polypeptides having diuretic andnatriuretic activities. Polypeptides having diuretic activity can beused medically to treat hypertension, kidney disease, cirrhosis,congestive heart failure, or any fluid overload state. Polypeptideshaving natriuretic activity can increase the removal of sodium from thebody and can be used medically to treat hypertension, kidney disease,cirrhosis, congestive heart failure, or any sodium overload state.

In some cases, a polypeptide provided herein can have diuretic andnatriuretic activities, while lacking the ability to lower bloodpressure. In some cases, a polypeptide provided herein can beadministered to a mammal having congestive heart failure underconditions that induce a detectable diuretic effect, without inducing adetectable natriuretic effect, and while affecting glomerular filtrationrate.

In general, one aspect of this document features a substantially purepolypeptide between 37 and 47 amino acid residues in length, wherein thepolypeptide comprises, or consists essentially of, an amino acidsequence (a) set forth in SEQ ID NO:1 or (b) that aligns to the sequenceset forth in SEQ ID NO:1 with five or less amino acid additions,deletions, substitutions, or combinations thereof. The length of thepolypeptide can be between 38 and 46 amino acid residues. The length ofthe polypeptide can be between 39 and 45 amino acid residues. The lengthof the polypeptide can be between 40 and 44 amino acid residues. Thelength of the polypeptide can be between 41 and 43 amino acid residues.The length of the polypeptide can be 42 amino acid residues. The lengthof the polypeptide can be 37 amino acid residues. The length of thepolypeptide can be 47 amino acid residues. The amino acid sequence canbe the sequence set forth in SEQ ID NO:1. The amino acid sequence canalign to the sequence set forth in SEQ ID NO:1 with four or less aminoacid additions, deletions, substitutions, or combinations thereof. Theamino acid sequence can align to the sequence set forth in SEQ ID NO:1with three or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with two or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with one orless amino acid additions, deletions, substitutions, or combinationsthereof. The amino acid sequence can align to the sequence set forth inSEQ ID NO:1 with five or less amino acid additions. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with five orless amino acid deletions. The amino acid sequence can align to thesequence set forth in SEQ ID NO:1 with five or less amino acidsubstitutions. The length of the polypeptide can be 42 amino acidresidues, and wherein the amino acid sequence is the sequence set forthin SEQ ID NO:1. The polypeptide can have diuretic and natriureticactivity. The polypeptide can lack the ability to lower blood pressurein a mammal. The mammal can be a human or dog.

In another aspect, this document features a substantially purepolypeptide between 45 and 65 amino acid residues in length, wherein thepolypeptide comprises, or consists essentially of, a first amino acidsequence: (a) set forth in SEQ ID NO:1 or (b) that aligns to thesequence set forth in SEQ ID NO:1 with five or less amino aciddeletions, substitutions, or combinations thereof, and wherein thepolypeptide comprises a second amino acid sequence: (a) set forth in SEQID NO:2 or (b) that aligns to the sequence set forth in SEQ ID NO:2 with(i) five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or (ii) fifteen or less amino aciddeletions. The length of the polypeptide can be between 58 and 63 aminoacid residues. The length of the polypeptide can be 60 amino acidresidues. The length of the polypeptide can be 45 amino acid residues.The polypeptide can be 65 amino acid residues. The sequence of thepolypeptide can be the sequence set forth in SEQ ID NO:3. Thepolypeptide can have diuretic and natriuretic activity.

In another aspect, this document features an isolated nucleic acidencoding a polypeptide between 37 and 47 amino acid residues in length,wherein the polypeptide comprises, or consists essentially of, an aminoacid sequence (a) set forth in SEQ ID NO:1 or (b) that aligns to thesequence set forth in SEQ ID NO:1 with five or less amino acidadditions, deletions, substitutions, or combinations thereof. The lengthof the polypeptide can be between 38 and 46 amino acid residues. Thelength of the polypeptide can be between 39 and 45 amino acid residues.The length of the polypeptide can be between 40 and 44 amino acidresidues. The length of the polypeptide can be between 41 and 43 aminoacid residues. The length of the polypeptide can be 42 amino acidresidues. The length of the polypeptide can be 37 amino acid residues.The length of the polypeptide can be 47 amino acid residues. The aminoacid sequence can be the sequence set forth in SEQ ID NO:1. The aminoacid sequence can align to the sequence set forth in SEQ ID NO:1 withfour or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with three or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with two orless amino acid additions, deletions, substitutions, or combinationsthereof. The amino acid sequence can align to the sequence set forth inSEQ ID NO:1 with one or less amino acid additions, deletions,substitutions, or combinations thereof. The amino acid sequence canalign to the sequence set forth in SEQ ID NO:1 with five or less aminoacid additions. The amino acid sequence can align to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions. The aminoacid sequence can align to the sequence set forth in SEQ ID NO:1 withfive or less amino acid substitutions. The length of the polypeptide canbe 42 amino acid residues, and wherein the amino acid sequence is thesequence set forth in SEQ ID NO:1. The polypeptide can have diuretic andnatriuretic activity. The polypeptide can lack the ability to lowerblood pressure in a mammal. The mammal can be a human or dog.

In another aspect, this document features an isolated nucleic acidencoding a polypeptide between 45 and 65 amino acid residues in length,wherein the polypeptide comprises, or consists essentially of, a firstamino acid sequence: (a) set forth in SEQ ID NO:1 or (b) that aligns tothe sequence set forth in SEQ ID NO:1 with five or less amino aciddeletions, substitutions, or combinations thereof, and wherein thepolypeptide comprises a second amino acid sequence: (a) set forth in SEQID NO:2 or (b) that aligns to the sequence set forth in SEQ ID NO:2 with(i) five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or (ii) fifteen or less amino aciddeletions. The length of the polypeptide can be between 58 and 63 aminoacid residues. The length of the polypeptide can be 60 amino acidresidues. The length of the polypeptide can be 45 amino acid residues.The polypeptide can be 65 amino acid residues. The sequence of thepolypeptide can be the sequence set forth in SEQ ID NO:3. Thepolypeptide can have diuretic and natriuretic activity.

In another aspect, this document features a vector comprising a nucleicacid encoding a polypeptide between 37 and 47 amino acid residues inlength, wherein the polypeptide comprises, or consists essentially of,an amino acid sequence (a) set forth in SEQ ID NO:1 or (b) that alignsto the sequence set forth in SEQ ID NO:1 with five or less amino acidadditions, deletions, substitutions, or combinations thereof. The lengthof the polypeptide can be between 38 and 46 amino acid residues. Thelength of the polypeptide can be between 39 and 45 amino acid residues.The length of the polypeptide can be between 40 and 44 amino acidresidues. The length of the polypeptide can be between 41 and 43 aminoacid residues. The length of the polypeptide can be 42 amino acidresidues. The length of the polypeptide can be 37 amino acid residues.The length of the polypeptide can be 47 amino acid residues. The aminoacid sequence can be the sequence set forth in SEQ ID NO:1. The aminoacid sequence can align to the sequence set forth in SEQ ID NO:1 withfour or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with three or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with two orless amino acid additions, deletions, substitutions, or combinationsthereof. The amino acid sequence can align to the sequence set forth inSEQ ID NO:1 with one or less amino acid additions, deletions,substitutions, or combinations thereof. The amino acid sequence canalign to the sequence set forth in SEQ ID NO:1 with five or less aminoacid additions. The amino acid sequence can align to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions. The aminoacid sequence can align to the sequence set forth in SEQ ID NO:1 withfive or less amino acid substitutions. The length of the polypeptide canbe 42 amino acid residues, and wherein the amino acid sequence is thesequence set forth in SEQ ID NO:1. The polypeptide can have diuretic andnatriuretic activity. The polypeptide can lack the ability to lowerblood pressure in a mammal. The mammal can be a human or dog.

In another aspect, this document features a vector comprising a nucleicacid encoding a polypeptide between 45 and 65 amino acid residues inlength, wherein the polypeptide comprises, or consists essentially of, afirst amino acid sequence: (a) set forth in SEQ ID NO:1 or (b) thataligns to the sequence set forth in SEQ ID NO:1 with five or less aminoacid deletions, substitutions, or combinations thereof, and wherein thepolypeptide comprises a second amino acid sequence: (a) set forth in SEQID NO:2 or (b) that aligns to the sequence set forth in SEQ ID NO:2 with(i) five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or (ii) fifteen or less amino aciddeletions. The length of the polypeptide can be between 58 and 63 aminoacid residues. The length of the polypeptide can be 60 amino acidresidues. The length of the polypeptide can be 45 amino acid residues.The polypeptide can be 65 amino acid residues. The sequence of thepolypeptide can be the sequence set forth in SEQ ID NO:3. Thepolypeptide can have diuretic and natriuretic activity.

In another aspect, this document features a host cell comprising anucleic acid encoding a polypeptide between 37 and 47 amino acidresidues in length, wherein the polypeptide comprises, or consistsessentially of, an amino acid sequence (a) set forth in SEQ ID NO:1 or(b) that aligns to the sequence set forth in SEQ ID NO:1 with five orless amino acid additions, deletions, substitutions, or combinationsthereof. The length of the polypeptide can be between 38 and 46 aminoacid residues. The length of the polypeptide can be between 39 and 45amino acid residues. The length of the polypeptide can be between 40 and44 amino acid residues. The length of the polypeptide can be between 41and 43 amino acid residues. The length of the polypeptide can be 42amino acid residues. The length of the polypeptide can be 37 amino acidresidues. The length of the polypeptide can be 47 amino acid residues.The amino acid sequence can be the sequence set forth in SEQ ID NO:1.The amino acid sequence can align to the sequence set forth in SEQ IDNO:1 with four or less amino acid additions, deletions, substitutions,or combinations thereof. The amino acid sequence can align to thesequence set forth in SEQ ID NO:1 with three or less amino acidadditions, deletions, substitutions, or combinations thereof. The aminoacid sequence can align to the sequence set forth in SEQ ID NO:1 withtwo or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with one or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with five orless amino acid additions. The amino acid sequence can align to thesequence set forth in SEQ ID NO:1 with five or less amino aciddeletions. The amino acid sequence can align to the sequence set forthin SEQ ID NO:1 with five or less amino acid substitutions. The length ofthe polypeptide can be 42 amino acid residues, and wherein the aminoacid sequence is the sequence set forth in SEQ ID NO:1. The polypeptidecan have diuretic and natriuretic activity. The polypeptide can lack theability to lower blood pressure in a mammal. The mammal can be a humanor dog. The host cell can be a eukaryotic host cell.

In another aspect, this document features a host cell comprising anucleic acid encoding a polypeptide between 45 and 65 amino acidresidues in length, wherein the polypeptide comprises, or consistsessentially of, a first amino acid sequence: (a) set forth in SEQ IDNO:1 or (b) that aligns to the sequence set forth in SEQ ID NO:1 withfive or less amino acid deletions, substitutions, or combinationsthereof, and wherein the polypeptide comprises a second amino acidsequence: (a) set forth in SEQ ID NO:2 or (b) that aligns to thesequence set forth in SEQ ID NO:2 with (i) five or less amino acidadditions, substitutions, or combinations thereof provided that theaddition or substitution does not result in the presence of a cysteineresidue or (ii) fifteen or less amino acid deletions. The length of thepolypeptide can be between 58 and 63 amino acid residues. The length ofthe polypeptide can be 60 amino acid residues. The length of thepolypeptide can be 45 amino acid residues. The polypeptide can be 65amino acid residues. The sequence of the polypeptide can be the sequenceset forth in SEQ ID NO:3. The polypeptide can have diuretic andnatriuretic activity. The host cell can be a eukaryotic host cell.

In another aspect, this document features a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a polypeptidebetween 37 and 47 amino acid residues in length, wherein the polypeptidecomprises, or consists essentially of, an amino acid sequence (a) setforth in SEQ ID NO:1 or (b) that aligns to the sequence set forth in SEQID NO:1 with five or less amino acid additions, deletions,substitutions, or combinations thereof. The length of the polypeptidecan be between 38 and 46 amino acid residues. The length of thepolypeptide can be between 39 and 45 amino acid residues. The length ofthe polypeptide can be between 40 and 44 amino acid residues. The lengthof the polypeptide can be between 41 and 43 amino acid residues. Thelength of the polypeptide can be 42 amino acid residues. The length ofthe polypeptide can be 37 amino acid residues. The length of thepolypeptide can be 47 amino acid residues. The amino acid sequence canbe the sequence set forth in SEQ ID NO:1. The amino acid sequence canalign to the sequence set forth in SEQ ID NO:1 with four or less aminoacid additions, deletions, substitutions, or combinations thereof. Theamino acid sequence can align to the sequence set forth in SEQ ID NO:1with three or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with two or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with one orless amino acid additions, deletions, substitutions, or combinationsthereof. The amino acid sequence can align to the sequence set forth inSEQ ID NO:1 with five or less amino acid additions. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with five orless amino acid deletions. The amino acid sequence can align to thesequence set forth in SEQ ID NO:1 with five or less amino acidsubstitutions. The length of the polypeptide can be 42 amino acidresidues, and wherein the amino acid sequence is the sequence set forthin SEQ ID NO:1. The polypeptide can have diuretic and natriureticactivity. The polypeptide can lack the ability to lower blood pressurein a mammal. The mammal can be a human or dog.

In another aspect, this document features a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a polypeptidebetween 45 and 65 amino acid residues in length, wherein the polypeptidecomprises, or consists essentially of, a first amino acid sequence: (a)set forth in SEQ ID NO:1 or (b) that aligns to the sequence set forth inSEQ ID NO:1 with five or less amino acid deletions, substitutions, orcombinations thereof, and wherein the polypeptide comprises a secondamino acid sequence: (a) set forth in SEQ ID NO:2 or (b) that aligns tothe sequence set forth in SEQ ID NO:2 with (i) five or less amino acidadditions, substitutions, or combinations thereof provided that theaddition or substitution does not result in the presence of a cysteineresidue or (ii) fifteen or less amino acid deletions. The length of thepolypeptide can be between 58 and 63 amino acid residues. The length ofthe polypeptide can be 60 amino acid residues. The length of thepolypeptide can be 45 amino acid residues. The polypeptide can be 65amino acid residues. The sequence of the polypeptide can be the sequenceset forth in SEQ ID NO:3. The polypeptide can have diuretic andnatriuretic activity.

In another aspect, this document features a method for increasingdiuretic and natriuretic activity within a mammal without lowering bloodpressure. The method comprises, or consists essentially of,administering a polypeptide to the mammal, wherein the polypeptide isbetween 37 and 47 amino acid residues in length, wherein the polypeptidecomprises, or consists essentially of, an amino acid sequence (a) setforth in SEQ ID NO:1 or (b) that aligns to the sequence set forth in SEQID NO:1 with five or less amino acid additions, deletions,substitutions, or combinations thereof. The length of the polypeptidecan be between 38 and 46 amino acid residues. The length of thepolypeptide can be between 39 and 45 amino acid residues. The length ofthe polypeptide can be between 40 and 44 amino acid residues. The lengthof the polypeptide can be between 41 and 43 amino acid residues. Thelength of the polypeptide can be 42 amino acid residues. The length ofthe polypeptide can be 37 amino acid residues. The length of thepolypeptide can be 47 amino acid residues. The amino acid sequence canbe the sequence set forth in SEQ ID NO:1. The amino acid sequence canalign to the sequence set forth in SEQ ID NO:1 with four or less aminoacid additions, deletions, substitutions, or combinations thereof. Theamino acid sequence can align to the sequence set forth in SEQ ID NO:1with three or less amino acid additions, deletions, substitutions, orcombinations thereof. The amino acid sequence can align to the sequenceset forth in SEQ ID NO:1 with two or less amino acid additions,deletions, substitutions, or combinations thereof. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with one orless amino acid additions, deletions, substitutions, or combinationsthereof. The amino acid sequence can align to the sequence set forth inSEQ ID NO:1 with five or less amino acid additions. The amino acidsequence can align to the sequence set forth in SEQ ID NO:1 with five orless amino acid deletions. The amino acid sequence can align to thesequence set forth in SEQ ID NO:1 with five or less amino acidsubstitutions. The length of the polypeptide can be 42 amino acidresidues, and wherein the amino acid sequence is the sequence set forthin SEQ ID NO:1. The polypeptide can have diuretic and natriureticactivity. The polypeptide can lack the ability to lower blood pressurein a mammal. The mammal can be a human or dog.

In another aspect, this document features a method for increasingdiuretic and natriuretic activity within a mammal without lowering bloodpressure. The method comprises, or consist essentially of, administeringa polypeptide to the mammal, wherein the polypeptide is between 45 and65 amino acid residues in length, wherein the polypeptide comprises, orconsists essentially of, a first amino acid sequence: (a) set forth inSEQ ID NO:1 or (b) that aligns to the sequence set forth in SEQ ID NO:1with five or less amino acid deletions, substitutions, or combinationsthereof, and wherein the polypeptide comprises a second amino acidsequence: (a) set forth in SEQ ID NO:2 or (b) that aligns to thesequence set forth in SEQ ID NO:2 with (i) five or less amino acidadditions, substitutions, or combinations thereof provided that theaddition or substitution does not result in the presence of a cysteineresidue or (ii) fifteen or less amino acid deletions. The length of thepolypeptide can be between 58 and 63 amino acid residues. The length ofthe polypeptide can be 60 amino acid residues. The length of thepolypeptide can be 45 amino acid residues. The polypeptide can be 65amino acid residues. The sequence of the polypeptide can be the sequenceset forth in SEQ ID NO:3. The polypeptide can have diuretic andnatriuretic activity.

In another aspect, this document features a method for treating a mammalhaving a renal dysfunction. The method comprises, or consistsessentially of, administering, to the mammal, a polypeptide underconditions wherein the severity of a symptom of the renal dysfunction isreduced. The mammal can be a human. The renal dysfunction can compriserenal failure. The renal dysfunction can comprise renal failureaccompanied with congestive heart failure. The polypeptide can beadministered intravenously, orally, or intranasally. The polypeptide canbe administered in a slow release formulation. The polypeptide can bebetween 37 and 47 amino acid residues in length and comprise an aminoacid sequence set forth in SEQ ID NO:1. The polypeptide can be between37 and 47 amino acid residues in length and comprises an amino acidsequence that aligns to the sequence set forth in SEQ ID NO:1 with fiveor less amino acid additions, deletions, substitutions, or combinationsthereof. The polypeptide can be between 45 and 65 amino acid residues inlength and comprise (i) a first amino acid sequence set forth in SEQ IDNO:1 and (ii) a second amino acid sequence set forth in SEQ ID NO:2. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence set forth in SEQ ID NO:2. The polypeptide can be between 45 and65 amino acid residues in length, comprises a first amino acid sequenceset forth in SEQ ID NO:1, and comprise a second amino acid sequence thataligns to the sequence set forth in SEQ ID NO:2 with (i) five or lessamino acid additions, substitutions, or combinations thereof providedthat the addition or substitution does not result in the presence of acysteine residue or (ii) fifteen or less amino acid deletions. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence that aligns to the sequence set forth in SEQ ID NO:2 with (i)five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or with (ii) fifteen or less aminoacid deletions. The symptom can comprise an abnormal serum creatininelevel, urine flow, renin level, glomerular filtration rate, urinary cGMPexcretion rate, urinary ANP excretion rate, urinary BNP excretion rate,cardiac output, systemic vascular resistance, or aldosterone level. Thesymptom can comprise reduced urine flow, and wherein the urine flow ofthe mammal increases at least 50% after the administration step. Thesymptom can comprise reduced renin level, and wherein the renin level ofthe mammal increases at least 50% after the administration step. Thesymptom can comprise reduced glomerular filtration rate, and wherein theglomerular filtration rate of the mammal increases at least 50% afterthe administration step. The symptom can comprise reduced urinary cGMPexcretion rate, and wherein the urinary cGMP excretion rate of themammal increases at least 25% after the administration step. The symptomcan comprise reduced urinary ANP excretion rate, and wherein the urinaryANP excretion rate of the mammal increases at least 25% after theadministration step. The symptom can comprise reduced urinary BNPexcretion rate, and wherein the urinary BNP excretion rate of the mammalincreases at least 25% after the administration step. The symptom cancomprise increased cardiac output, and wherein the cardiac output of themammal decreased at least 2% after the administration step. The symptomcan comprise reduced systemic vascular resistance, and wherein thesystemic vascular resistance of the mammal increases at least 10% afterthe administration step. The symptom can comprise reduced aldosteronelevel, and wherein the aldosterone level of the mammal increases atleast 10% after the administration step.

In another aspect, this document features a method for treating a mammalhaving an inflammatory condition. The method comprises, or consistsessentially of, administering, to the mammal, a polypeptide underconditions wherein the severity of a symptom of the inflammatorycondition is reduced. The mammal can be a human. The polypeptide can beadministered intravenously, orally, or intranasally. The polypeptide canbe administered in a slow release formulation. The polypeptide can bebetween 37 and 47 amino acid residues in length and comprise an aminoacid sequence set forth in SEQ ID NO:1. The polypeptide can be between37 and 47 amino acid residues in length and comprise an amino acidsequence that aligns to the sequence set forth in SEQ ID NO:1 with fiveor less amino acid additions, deletions, substitutions, or combinationsthereof. The polypeptide can be between 45 and 65 amino acid residues inlength and comprise (i) a first amino acid sequence set forth in SEQ IDNO:1 and (ii) a second amino acid sequence set forth in SEQ ID NO:2. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence set forth in SEQ ID NO:2. The polypeptide can be between 45 and65 amino acid residues in length, comprise a first amino acid sequenceset forth in SEQ ID NO:1, and comprise a second amino acid sequence thataligns to the sequence set forth in SEQ ID NO:2 with (i) five or lessamino acid additions, substitutions, or combinations thereof providedthat the addition or substitution does not result in the presence of acysteine residue or (ii) fifteen or less amino acid deletions. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence that aligns to the sequence set forth in SEQ ID NO:2 with (i)five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or with (ii) fifteen or less aminoacid deletions.

In another aspect, this document features a method for treating a mammalhaving a heart dysfunction. The method comprises, or consistsessentially of, administering, to the mammal, a polypeptide underconditions wherein the severity of a symptom of the heart dysfunction isreduced. The mammal can be a human. The heart dysfunction can compriseheart failure. The heart dysfunction can comprise congestive heartfailure accompanied with renal failure. The polypeptide can beadministered intravenously, orally, or intranasally. The polypeptide canbe administered in a slow release formulation. The polypeptide can bebetween 37 and 47 amino acid residues in length and comprise an aminoacid sequence set forth in SEQ ID NO:1. The polypeptide can be between37 and 47 amino acid residues in length and comprise an amino acidsequence that aligns to the sequence set forth in SEQ ID NO:1 with fiveor less amino acid additions, deletions, substitutions, or combinationsthereof. The polypeptide can be between 45 and 65 amino acid residues inlength and comprise (i) a first amino acid sequence set forth in SEQ IDNO:1 and (ii) a second amino acid sequence set forth in SEQ ID NO:2. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence set forth in SEQ ID NO:2. The polypeptide can be between 45 and65 amino acid residues in length, comprise a first amino acid sequenceset forth in SEQ ID NO:1, and comprise a second amino acid sequence thataligns to the sequence set forth in SEQ ID NO:2 with (i) five or lessamino acid additions, substitutions, or combinations thereof providedthat the addition or substitution does not result in the presence of acysteine residue or (ii) fifteen or less amino acid deletions. Thepolypeptide can be between 45 and 65 amino acid residues in length,comprise a first amino acid sequence that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid deletions,substitutions, or combinations thereof, and comprise a second amino acidsequence that aligns to the sequence set forth in SEQ ID NO:2 with (i)five or less amino acid additions, substitutions, or combinationsthereof provided that the addition or substitution does not result inthe presence of a cysteine residue or with (ii) fifteen or less aminoacid deletions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of an ASBNP polypeptide that is 60 aminoacid residues in length (SEQ ID NO:4), an ASBNP.1 polypeptide that is 42amino acid residues in length (SEQ ID NO:1), an ASBNP.2 polypeptide thatis 60 amino acid residues in length with an alanine at position 43 (SEQID NO:3). The sequence of ASBNP.2 from the alanine at position 43 to theleucine at position 60 is 18 amino acid residues in length (SEQ IDNO:2). ASBNP (also referred to as BNP2) is a variant form of BNPgenerated by alternative splicing.

FIG. 2 is a bar graph plotting urine flow rates for dogs treated withASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 3 is a bar graph plotting urinary sodium excretion rates for dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 4 is a bar graph plotting distal fractional tubular sodiumreabsorption rates for dogs treated with ASBNP.1 as indicated. Baselineis prior to administration; low is 2 pmol; high is 10 pmol; super highis 100 pmol; rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 5 is a bar graph plotting proximal tubular fractional sodiumreabsorption rates for dogs treated with ASBNP.1 as indicated. Baselineis prior to administration; low is 2 pmol; high is 10 pmol; super highis 100 pmol; and rec 1 is recovery 1.

FIG. 6 is a bar graph plotting plasma cGMP levels for dogs treated withASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 7 is a bar graph plotting plasma renin activity for dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 8 is a bar graph plotting glomerular filtration rates for dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 9 is a bar graph plotting renal blood flow rates for dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 10 is a bar graph plotting mean arterial blood pressure levels fordogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 11 is a bar graph plotting cGMP levels in HUVECS treated with BNP,ASBNP, or ASBNP.1 at the indicated concentrations. OX representsoxidized, and non-OX represents non-oxidized.

FIG. 12 is a bar graph plotting cGMP levels in HASMCS treated with BNP,ASBNP, or ASBNP.1 at the indicated concentrations. OX representsoxidized, and non-OX represents non-oxidized.

FIG. 13 is a line graph plotting vasoactivity measurements obtained fromrabbit vascular rings treated with either BNP, ASBNP, or ASBNP.1 at theindicated concentrations.

FIG. 14 contains a nucleic acid sequence (SEQ ID NO:5) that can encodean ASBNP.1 polypeptide and a nucleic acid sequence (SEQ ID NO:6) thatcan encode an ASBNP.2 polypeptide.

FIG. 15 is a bar graph plotting urine flow rates for paced dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 16 is a bar graph plotting urinary sodium excretion rates for paceddogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 17 is a bar graph plotting distal fractional tubular sodiumreabsorption rates for paced dogs treated with ASBNP.1 as indicated.Baseline is prior to administration; low is 2 pmol; high is 10 pmol;super high is 100 pmol; rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 18 is a bar graph plotting proximal tubular fractional sodiumreabsorption rates for paced dogs treated with ASBNP.1 as indicated.Baseline is prior to administration; low is 2 pmol; high is 10 pmol;super high is 100 pmol; rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 19 is a bar graph plotting plasma cGMP levels for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 20 is a bar graph plotting plasma renin activity (ng/mL/hour) forpaced dogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 21 is a bar graph plotting glomerular filtration rates for paceddogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 22 is a bar graph plotting renal blood flow rates for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 23 is a bar graph plotting mean arterial blood pressure levels forpaced dogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 24 is a bar graph plotting urinary cGMP excretion for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 25 is a bar graph plotting urinary ANP excretion for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 26 is a bar graph plotting urinary BNP excretion for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 27 is a bar graph plotting plasma BNP levels for paced dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 28 is a bar graph plotting plasma ANP levels for paced dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 29 is a bar graph plotting pulmonary capillary wedge pressure forpaced dogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol; high is 10 pmol; super high is 100 pmol;rec 1 is recovery 1; and rec 2 is recovery 2.

FIG. 30 is a bar graph plotting cardiac output for paced dogs treatedwith ASBNP.1 as indicated. Baseline is prior to administration; low is 2pmol; high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; andrec 2 is recovery 2.

FIG. 31 is a bar graph plotting systemic vascular resistance for paceddogs treated with ASBNP.1 as indicated. Baseline is prior toadministration; low is 2 pmol;

high is 10 pmol; super high is 100 pmol; rec 1 is recovery 1; and rec 2is recovery 2.

FIG. 32 is a bar graph plotting angiotensin II levels for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 33 is a bar graph plotting aldosterone levels for paced dogstreated with ASBNP.1 as indicated. Baseline is prior to administration;low is 2 pmol; high is 10 pmol; super high is 100 pmol; rec 1 isrecovery 1; and rec 2 is recovery 2.

FIG. 34 is a graph plotting urinary sodium excretion (microEq/min) inpaced dogs at the indicated times after beginning administration ofASBNP.1 polypeptide (100 pmol/kg/minute) for 90 minutes. BL=baseline; 30min=30 minutes of ASBNP.1 polypeptide administration; 60 min=60 minutesof ASBNP.1 polypeptide administration; 90 min=90 minutes of ASBNP.1polypeptide administration; Washout=after washout period at stop ofinfusion; Rec 1=30 minutes after stopping infusion; and Rec 2=60 minutesafter stopping infusion.

FIG. 35 is a graph plotting urine flow (mL/min) in paced dogs at theindicated times after beginning administration of ASBNP.1 polypeptide(100 pmol/kg/minute) for 90 minutes. BL=baseline; 30 min=30 minutes ofASBNP.1 polypeptide administration; 60 min=60 minutes of ASBNP.1polypeptide administration; 90 min=90 minutes of ASBNP.1 polypeptideadministration; Washout=after washout period at stop of infusion; Rec1=30 minutes after stopping infusion; and Rec 2=60 minutes afterstopping infusion.

FIG. 36 is a graph plotting mean arterial blood pressure (mmHg) in paceddogs at the indicated times after beginning administration of ASBNP.1polypeptide (100 pmol/kg/minute) for 90 minutes. BL=baseline; 30 min=30minutes of ASBNP.1 polypeptide administration; 60 min=60 minutes ofASBNP.1 polypeptide administration; 90 min=90 minutes of ASBNP.1polypeptide administration; Washout=after washout period at stop ofinfusion; Rec 1=30 minutes after stopping infusion; and Rec 2=60 minutesafter stopping infusion.

FIG. 37 is a graph plotting renal blood flow in paced dogs at theindicated times after beginning administration of ASBNP.1 polypeptide(100 pmol/kg/minute) for 90 minutes. BL=baseline; 30 min=30 minutes ofASBNP.1 polypeptide administration; 60 min=60 minutes of ASBNP.1polypeptide administration; 90 min=90 minutes of ASBNP.1 polypeptideadministration; Washout=after washout period at stop of infusion; Rec1=30 minutes after stopping infusion; and Rec 2=60 minutes afterstopping infusion.

FIG. 38 is a graph plotting pulmonary capillary wedge pressure in paceddogs at the indicated times after beginning administration of ASBNP.1polypeptide (100 pmol/kg/minute) for 90 minutes. BL=baseline; 30 min=30minutes of ASBNP.1 polypeptide administration; 60 min=60 minutes ofASBNP.1 polypeptide administration; 90 min=90 minutes of ASBNP.1polypeptide administration; Washout=after washout period at stop ofinfusion; Rec 1=30 minutes after stopping infusion; and Rec 2=60 minutesafter stopping infusion.

FIG. 39 is a graph plotting cardiac output (1/min) in paced dogs at theindicated times after beginning administration of ASBNP.1 polypeptide(100 pmol/kg/minute) for 90 minutes. BL=baseline; 30 min=30 minutes ofASBNP.1 polypeptide administration; 60 min=60 minutes of ASBNP.1polypeptide administration; 90 min=90 minutes of ASBNP.1 polypeptideadministration; Washout=after washout period at stop of infusion; Rec1=30 minutes after stopping infusion; and Rec 2=60 minutes afterstopping infusion.

DETAILED DESCRIPTION

This document relates to diuretic and natriuretic polypeptides. Forexample, this document provides polypeptides having diuretic and/ornatriuretic activities. In some cases, a polypeptide provided herein canhave diuretic and/or natriuretic activities, while lacking the abilityto lower blood pressure. This document also provides methods andmaterials for inducing diuretic and/or natriuretic activities within amammal.

A polypeptide provided herein can have any sequence and can have anylength. For example, a polypeptide provided herein can include thesequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In somecases, a polypeptide provided herein can contain an amino acid sequencethat aligns to the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3 with ten or less (e.g., nine or less, eight or less, sevenor less, six or less, five or less, four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:1 with the exception thatfirst serine residue or the last valine residue of SEQ ID NO:1 isdeleted or replaced with a different amino acid residue.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:1 or aligns tothe sequence set forth in SEQ ID NO:1 with ten or less (e.g., nine orless, eight or less, seven or less, six or less, five or less, four orless, three or less, two or less, one, or zero) amino acid deletions,substitutions, or combinations thereof and (b) a second amino acidsequence that either is set forth in SEQ ID NO:2 or aligns to thesequence set forth in SEQ ID NO:2 with either (i) ten or less (e.g.,nine or less, eight or less, seven or less, six or less, five or less,four or less, three or less, two or less, one, or zero) amino acidadditions, substitutions, or combinations thereof provided that theaddition or substitution does not result in the presence of a cysteineresidue or (ii) 15 or less (e.g., 14 or less, 13 or less, twelve orless, eleven or less, ten or less, nine or less, eight or less, seven orless, six or less, five or less, four or less, three or less, two orless, one, or zero) amino acid deletions. For example, a polypeptideprovided herein can comprise or consist of the sequence set forth in SEQID NO:3 with the exception that the cysteine residue at position 43 ofSEQ ID NO:3 is an amino acid other than cysteine (e.g., alanine,arginine, asparagines, aspartic acid, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,praline, serine, threonine, tryptophan, tyrosine, or valine).

Polypeptides having one or more amino acid substitutions relative to apolypeptide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3 can be prepared and modified as described herein. Amino acidsubstitutions can be conservative or non-conservative amino acidsubstitutions. Conservative amino acid substitutions include, forexample, substitution of an acidic amino acid residue (e.g., asparticacid or glutamic acid) with another acidic amino acid residue,substitution of a basic amino acid residue (e.g., lysine, arginine, orhistidine) with another basic amino acid residue, substitution of ahydrophobic amino acid residue with another hydrophobic amino acidresidue (e.g., substitution of leucine with isoleucine, methionine withvaline, or alanine with valine), and substitution of a hydrophilic aminoacid residue (e.g., serine, glycine, or threonine) with anotherhydrophilic amino acid residue.

Conservative amino acid substitutions also include substitution of anamino acid residue having a particular type of side chain with anotheramino acid residue having a similar type of side chain. For example,conservative amino acid substitutions include substitution of an aminoacid residue having an aliphatic side chain (e.g., glycine, alanine,valine, leucine, or isoleucine) with another amino acid residue havingan aliphatic side chain, substitution of an amino acid residue having analiphatic-hydroxyl side chain (e.g., serine or threonine) with anotheramino acid residue having an aliphatic-hydroxyl side chain, substitutionof an amino acid residue having an amide-containing side chain (e.g.,asparagine or glutamine) with another amino acid residue having anamide-containing side chain, substitution of an amino acid residuehaving an aromatic side chain (e.g., phenylalanine, tyrosine, ortryptophan) with another amino acid residue having an aromatic sidechain, substitution of an amino acid residue having a basic side chain(e.g., lysine, arginine, or histidine) with another amino acid residuehaving a basic side chain, and substitution of an amino acid residuehaving a sulfur-containing side chain (e.g., cysteine or methionine)with another amino acid residue having a sulfur-containing side chain.

A polypeptide provided herein can have any length. For example, apolypeptide provided herein can be between 25 and 75 (e.g., between 30and 70, between 32 and 60, between 32 and 57, between 32 and 50, between32 and 45, between 35 and 43, or between 38 and 43) amino acid residuesin length. It will be appreciated that a polypeptide with a length of 25or 75 amino acid residues is a polypeptide with a length between 25 and75 amino acid residues.

In some cases, a polypeptide provided herein can be between 37 and 47amino acid residues in length and can comprise an amino acid sequence(a) set forth in SEQ ID NO:1 or (b) that aligns to the sequence setforth in SEQ ID NO:1 with five or less amino acid additions, deletions,substitutions, or combinations thereof. An example of such a polypeptideincludes, without limitation, an ASBNP.1 polypeptide. In some cases, apolypeptide provided herein can be between 45 and 65 amino acid residuesin length and can comprise (a) a first amino acid sequence that eitheris set forth in SEQ ID NO:1 or aligns to the sequence set forth in SEQID NO:1 with five or less amino acid deletions, substitutions, orcombinations thereof and (b) a second amino acid sequence that either isset forth in SEQ ID NO:2 or aligns to the sequence set forth in SEQ IDNO:2 with (i) five or less amino acid additions, substitutions, orcombinations thereof provided that the addition or substitution does notresult in the presence of a cysteine residue or (ii) fifteen or lessamino acid deletions. An example of such a polypeptide includes, withoutlimitation, an ASBNP.2 polypeptide.

In some cases, a polypeptide provided herein can be a substantially purepolypeptide. As used herein, the term “substantially pure” withreference to a polypeptide means that the polypeptide is substantiallyfree of other polypeptides, lipids, carbohydrates, and nucleic acid withwhich it is naturally associated. Thus, a substantially pure polypeptideis any polypeptide that is removed from its natural environment and isat least 60 percent pure or is any chemically synthesized polypeptide. Asubstantially pure polypeptide can be at least about 60, 65, 70, 75, 80,85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

In some embodiments, a polypeptide provide herein can lack the abilityto stimulate production of cGMP in human umbilical vascular endothelialcells (HUVEC). Intracellular cGMP production can be assayed using, forexample, the BIOTRACK cGMP enzyme immunoassay kit (Amersham PharmaciaBiotech). In other embodiments, a polypeptide provide herein can lackvasoactivity. Vasoactivity can be assessed by determining responsitivityof a blood vessel (e.g., a carotid artery in an organ chamber) to thepolypeptide.

A polypeptide provide herein can be obtained by expression of arecombinant nucleic acid encoding the polypeptide or by chemicalsynthesis. For example, standard recombinant technology using expressionvectors encoding a polypeptide provide herein can be used. The resultingpolypeptides then can be purified using, for example, affinitychromatographic techniques and HPLC. The extent of purification can bemeasured by any appropriate method, including but not limited to: columnchromatography, polyacrylamide gel electrophoresis, or high-performanceliquid chromatography. A polypeptide provide herein can be designed orengineered to contain a tag sequence that allows the polypeptide to bepurified (e.g., captured onto an affinity matrix). For example, a tagsuch as c-myc, hemagglutinin, polyhistidine, or Flag™ tag (Kodak) can beused to aid polypeptide purification. Such tags can be inserted anywherewithin the polypeptide including at either the carboxyl or aminotermini. Other fusions that can be used include enzymes that aid in thedetection of the polypeptide, such as alkaline phosphatase.

A polypeptide provided herein can be produced that contains two regions,a first region that includes the N-terminus and ring structure of amature natriuretic polypeptide (e.g., BNP, DNP, ANP, or CNP) and asecond region that includes a mutated or truncated version of theC-terminal portion of ASBNP. The N-termini and ring structures of BNP,DNP, ANP, and CNP are described elsewhere. See, e.g., U.S. patentapplication Ser. No. 10/561,014.

A polypeptide provided herein can be formulated as a pharmaceuticalcomposition by admixture with pharmaceutically acceptable non-toxicexcipients or carriers. Such compositions can be administered to asubject in need thereof in an amount effective to treat, for example,heart, liver, kidney, or other sodium retaining conditions. For example,such compositions can be administered to a subject having a renaldysfunction. A renal dysfunction can include, without limitation, acuterenal failure, glomerulonephritis, chronic renal failure, azotemia,uremia, immune renal disease; acute nephritic syndrome, rapidlyprogressive nephritic syndrome, nephrotic syndrome, Berger's Disease,chronic nephritic/proteinuric syndrome, tubulointerstital disease,nephrotoxic disorders, renal infarction, atheroembolic renal disease,renal cortical necrosis, malignant nephroangiosclerosis, renal veinthrombosis, renal tubular acidosis, renal glucosuria, nephrogenicdiabetes insipidus, Bartter's Syndrome, Liddle's Syndrome, polycysticrenal disease, interstitial nephritis, acute hemolytic uremic syndrome,medullary cystic disease, medullary sponge kidney, hereditary nephritis,and nail-patella syndrome.

Compositions provided herein also can be administered to a subjecthaving a heart dysfunction. A heart dysfunction can include, withoutlimitation, CHF, dilated congestive cardiomyopathy, hypertrophiccardiomyopathy, restrictive cardiomyopathy, mitral valve disease, aorticvalve disease, tricuspid valve disease, angina pectoris, myocardialinfarction, cardiac arrhythmia, pulmonary hypertension, arterialhypertension, renovascular hypertension, arteriosclerosis,atherosclerosis, and cardiac tumors.

Compositions provided herein also can be administered to a subjecthaving an inflammatory condition. An inflammatory condition can include,without limitation, myocarditis, asthma, chronic inflammation,autoimmune diabetes, tumor angiogenesis, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock,Gram-negative sepsis, toxic shock syndrome, asthma, adult respiratorydistress syndrome, stroke, reperfusion injury, CNS injuries such asneural trauma and ischemia, psoriasis restenosis, cerebral malaria,chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis,bone resorption diseases such as osteoporosis, graft versus hostreaction, Crohn's Disease, ulcerative colitis including inflammatorybowel disease (IBD), and pyresis.

Pharmaceutical compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration maybe prepared as desired using standard methods.

Formulations for parenteral administration may contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and the like. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxethylene-polyoxypropylene copolymers are examples of excipientsfor controlling the release of the polypeptide in vivo. Other suitableparenteral delivery systems include ethylene-vinyl acetate copolymerparticles, osmotic pumps, implantable infusion systems, and liposomes.Formulations for inhalation administration may contain excipients suchas lactose, if desired. Inhalation formulations may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or they may be oily solutions for administration inthe form of nasal drops. If desired, the compounds can be formulated asgels to be applied intranasally. Formulations for parenteraladministration may also include glycocholate for buccal administration

For oral administration, tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g. magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). Tablets can be coated by methods known in the art.Preparations for oral administration can also be formulated to givecontrolled release of the compound.

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulised aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

Polypeptides described herein also can be formulated for topicaldelivery. Topical application and/or delivery of a polypeptide providedherein can be achieved using known methods, e.g., iontophoresis orlipogels.

Compositions described herein (e.g., including a polypeptide providedherein) can additionally include other active ingredients.

Various clinical parameters can be monitored prior to, during, and/orafter administering a polypeptide described herein (e.g., formulated asa pharmaceutical composition) to a subject (e.g., a subject having arenal or heart dysfunction or an inflammatory condition). For example,vital signs, electrolytes, serum creatinine, cystatin, urinary BNPlevels, plasma BNP levels, urine output, plasma levels of theadministered polypeptide, urine levels of the administered polypeptide,or any combination thereof can be monitored. In some cases, plasma reninactivity, glomerular filtration rate, urinary cGMP excretion, plasmacGMP levels, urinary ANP excretion, urinary BNP excretion, cardiacoutput, systemic vascular resistance, aldosterone levels, or anycombination thereof can be monitored. Any appropriate method can be usedto monitor clinical parameters including, without limitation, themethods described herein.

Monitoring clinical parameters can allow a clinician to determinewhether or not an administered polypeptide is effective, e.g., whetheror not the severity of a symptom of a heart or renal dysfunction orinflammatory condition has been reduced. In addition, monitoringclinical parameters before, during, and/or after administration of apolypeptide provided herein can indicate whether the dose of thepolypeptide should be increased or decreased, whether administration ofthe polypeptide should be continued or discontinued, or whether thepolypeptide should be re-administered. Monitoring clinical parametersalso can indicate the severity of a subject's condition, which, in turn,can provide guidance as to when a polypeptide provided herein should beadministered and at what dose.

Nucleic Acids Encoding Polypeptides

This document also provides isolated nucleic acids that encode one ormore of the polypeptides provided herein. The term “isolated” as usedherein with reference to nucleic acid refers to a naturally-occurringnucleic acid that is not immediately contiguous with both of thesequences with which it is immediately contiguous (one on the 5′ end andone on the 3′ end) in the naturally-occurring genome of the organismfrom which it is derived. For example, an isolated nucleic acid can be,without limitation, a recombinant DNA molecule of any length, providedone of the nucleic acid sequences normally found immediately flankingthat recombinant DNA molecule in a naturally-occurring genome is removedor absent. Thus, an isolated nucleic acid includes, without limitation,a recombinant DNA that exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences as well as recombinant DNAthat is incorporated into a vector, an autonomously replicating plasmid,a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include a recombinant DNA molecule that is part of ahybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid (e.g., a nucleic acid encoding apolypeptide comprising or consisting of the amino acid sequence setforth in SEQ ID NO:1) can be made using common molecular cloning orchemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

As used herein, the term “nucleic acid” refers to both RNA and DNA,including mRNA, cDNA, genomic DNA, synthetic (e.g., chemicallysynthesized) DNA, and nucleic acid analogs. The nucleic acid can bedouble-stranded or single-stranded, and where single-stranded, can bethe sense strand or the antisense strand. In addition, nucleic acid canbe circular or linear. Nucleic acid analogs can be modified at the basemoiety, sugar moiety, or phosphate backbone to improve, for example,stability, hybridization, or solubility of a nucleic acid. Modificationsat the base moiety include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. Modifications of the sugar moiety can includemodification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methylor 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modifiedto produce morpholino nucleic acids, in which each base moiety is linkedto a six-membered, morpholino ring, or peptide nucleic acids, in whichthe deoxyphosphate backbone is replaced by a pseudopeptide backbone andthe four bases are retained. See, for example, Summerton and WellerAntisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup et al.Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphatebackbone can be replaced with, for example, a phosphorothioate orphosphorodithioate backbone, a phosphoroamidite, or an alkylphosphotriester backbone.

A nucleic acid provided herein can comprise or consist of the sequenceset forth in SEQ ID NO:5 or 6.

Typically, an isolated nucleic acid provided herein is at least 10nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,200, 300, 350, 400, or more nucleotides in length). Nucleic acidmolecules that are less than full-length can be useful, for example, asprimers or probes for diagnostic purposes. Isolated nucleic acidmolecules can be produced by standard techniques, including, withoutlimitation, common molecular cloning and chemical nucleic acid synthesistechniques. For example, polymerase chain reaction (PCR) techniques canbe used. PCR refers to a procedure or technique in which target nucleicacids are enzymatically amplified. Sequence information from the ends ofthe region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers typically are 15 to 50nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. For example, a primer can be 12, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45nucleotides in length. A primer can be purified from a restrictiondigest by conventional methods, or can be chemically synthesized.Primers typically are single-stranded for maximum efficiency inamplification, but a primer can be double-stranded. Double-strandedprimers are first denatured (e.g., treated with heat) to separate thestrands before use in amplification. General PCR techniques aredescribed, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids as described elsewhere (Lewis,Genetic Engineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl.Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292(1991)).

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids also can be obtained by mutagenesis. For example,a nucleic acid sequence encoding a polypeptide having the sequence setforth in SEQ ID NO:1, 2, or 3 can be mutated using standard techniquessuch as, for example, oligonucleotide-directed mutagenesis and/orsite-directed mutagenesis through PCR. See, Short Protocols in MolecularBiology, Chapter 8, Green Publishing Associates and John Wiley & Sons,Edited by Ausubel et al., 1992. Such mutations include additions,deletions, substitutions, and combinations thereof.

Vectors and Host Cells

This document also provides vectors containing a nucleic acid providedherein. As used herein, a “vector” is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. A vector can bean expression vector. An “expression vector” is a vector that includesone or more expression control sequences, and an “expression controlsequence” is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence.

In an expression vector provided herein, the nucleic acid can beoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 nucleotides upstream of the point atwhich transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it can be necessary to position the translation initiationsite of the translational reading frame of the polypeptide between oneand about fifty nucleotides downstream of the promoter. Enhancersprovide expression specificity in terms of time, location, and level.Unlike promoters, enhancers can function when located at variousdistances from the transcription site. An enhancer also can be locateddownstream from the transcription initiation site. A coding sequence is“operably linked” and “under the control” of expression controlsequences in a cell when RNA polymerase is able to transcribe the codingsequence into mRNA, which then can be translated into the polypeptideencoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,poxviruses, adenoviruses, and adeno-associated viruses. Numerous vectorsand expression systems are commercially available from such corporationsas Novagen (Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene(La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acidmolecule and/or nucleic acid vector provided herein. The term “hostcell” is intended to include prokaryotic and eukaryotic cells into whicha nucleic acid molecule or vector can be introduced. Any method can beused to introduce nucleic acid into a cell. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer can be usedintroduce nucleic acid into cells. In addition, naked DNA can bedelivered directly to cells in vivo as described elsewhere (U.S. Pat.Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document provides methods and materials for detecting a polypeptideprovided herein. Such methods and materials can be used to monitorpolypeptide levels within a mammal receiving the polypeptide as atherapeutic. The polypeptide provided herein (e.g., ASBNP.1 and ASBNP.2)can be detected, for example, immunologically using one or moreantibodies. As used herein, the term “antibody” includes intactmolecules as well as fragments thereof that are capable of binding to anepitopic determinant of a polypeptide provided herein. The term“epitope” refers to an antigenic determinant on an antigen to which theparatope of an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains, and typically have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.Epitopes generally have at least five contiguous amino acids (acontinuous epitope), or alternatively can be a set of noncontiguousamino acids that define a particular structure (e.g., a conformationalepitope). The term “antibody” includes polyclonal antibodies, monoclonalantibodies, humanized or chimeric antibodies, single chain Fv antibodyfragments, Fab fragments, and F(ab)₂ fragments. Polyclonal antibodiesare heterogenous populations of antibody molecules that are contained inthe sera of the immunized animals. Monoclonal antibodies are homogeneouspopulations of antibodies to a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a polypeptideprovided herein (e.g., ASBNP.1 and ASBNP.2) can be generated by knowntechniques. For example, F(ab′)2 fragments can be produced by pepsindigestion of the antibody molecule; Fab fragments can be generated byreducing the disulfide bridges of F(ab′)2 fragments. Alternatively, Fabexpression libraries can be constructed. See, for example, Huse et al.,Science, 246:1275 (1989). Once produced, antibodies or fragments thereofare tested for recognition of a polypeptide provided herein by standardimmunoassay methods including ELISA techniques, radioimmunoassays, andWestern blotting. See, Short Protocols in Molecular Biology, Chapter 11,Green Publishing Associates and John Wiley & Sons, Edited by Ausubel, F.M et al., 1992.

In immunological assays, an antibody having specific binding affinityfor a polypeptide provided herein or a secondary antibody that binds tosuch an antibody can be labeled, either directly or indirectly. Suitablelabels include, without limitation, radionuclides (e.g. ¹²⁵I, ¹³¹I, ³⁵S,³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, FITC,PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.), compounds that absorb light of a defined wavelength, or enzymes(e.g., alkaline phosphatase or horseradish peroxidase). Antibodies canbe indirectly labeled by conjugation with biotin then detected withavidin or streptavidin labeled with a molecule described above. Methodsof detecting or quantifying a label depend on the nature of the labeland are known in the art. Examples of detectors include, withoutlimitation, x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting a polypeptide provided herein can beperformed in a variety of known formats, including sandwich assays,competition assays (competitive RIA), or bridge immunoassays. See, forexample, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074.Methods of detecting a polypeptide provided herein generally includecontacting a biological sample with an antibody that binds to apolypeptide provided herein and detecting binding of the polypeptide tothe antibody. For example, an antibody having specific binding affinityfor a polypeptide provided herein can be immobilized on a solidsubstrate by any of a variety of methods known in the art and thenexposed to the biological sample. Binding of the polypeptide to theantibody on the solid substrate can be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).Alternatively, the antibody is labeled and detected as described above.A standard curve using known quantities of a polypeptide provided hereincan be generated to aid in the quantitation of the levels of thepolypeptide.

In other embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate is used to detect the presence,absence, or level of a polypeptide provided herein. The solid substratecan be contacted with the biological sample such that any polypeptide ofinterest in the sample can bind to the immobilized antibody. Thepresence, absence, or level of the polypeptide bound to the antibody canbe determined using a “detection” antibody having specific bindingaffinity for the polypeptide. In some embodiments, a capture antibodycan be used that has binding affinity for BNP as well as a polypeptideprovided herein. In this embodiment, a detection antibody can be usedthat has specific binding affinity for a particular polypeptide providedherein. It is understood that in sandwich assays, the capture antibodyshould not bind to the same epitope (or range of epitopes in the case ofa polyclonal antibody) as the detection antibody. Thus, if a monoclonalantibody is used as a capture antibody, the detection antibody can beanother monoclonal antibody that binds to an epitope that is eithercompletely physically separated from or only partially overlaps with theepitope to which the capture monoclonal antibody binds, or a polyclonalantibody that binds to epitopes other than or in addition to that towhich the capture monoclonal antibody binds. If a polyclonal antibody isused as a capture antibody, the detection antibody can be either amonoclonal antibody that binds to an epitope that is either completelyphysically separated from or partially overlaps with any of the epitopesto which the capture polyclonal antibody binds, or a polyclonal antibodythat binds to epitopes other than or in addition to that to which thecapture polyclonal antibody binds. Sandwich assays can be performed assandwich ELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Antibodies having specific binding affinity for a polypeptide providedherein can be produced through standard methods. In general, apolypeptide can be recombinantly produced as described above, or can bepurified from a biological sample (e.g., a heterologous expressionsystem), and used to immunize host animals, including rabbits, chickens,mice, guinea pigs, or rats. For example, a polypeptide having the aminoacid sequence set forth in SEQ ID NOs:1 or 2, or fragments thereof thatare at least six amino acids in length, can be used to immunize ananimal. Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin anddinitrophenol. Monoclonal antibodies can be prepared using a polypeptideprovided herein and standard hybridoma technology. In particular,monoclonal antibodies can be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines in culturesuch as described by Kohler et al., Nature, 256:495 (1975), the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72(1983); Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026 (1983)), andthe EBV-hybridoma technique (Cole et al., “Monoclonal Antibodies andCancer Therapy,” Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodiescan be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD,and any subclass thereof. The hybridoma producing the monoclonalantibodies can be cultivated in vitro and in vivo.

Alternative techniques for detecting a polypeptide provided hereininclude mass-spectrophotometric techniques such as electrosprayionization (ESI), and matrix-assisted laser desorption-ionization(MALDI). See, for example, Gevaert et al., Electrophoresis,22(9):1645-51 (2001); Chaurand et al., J. Am. Soc. Mass Spectrom.,10(2):91-103 (1999). Mass spectrometers useful for such applications areavailable from Applied Biosystems (Foster City, Calif.); BrukerDaltronics (Billerica, Mass.); and Amersham Pharmacia (Sunnyvale,Calif.).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Biological Effects of ASBNP.1 Polypeptides

A truncated form of ASBNP that terminates prior to the cysteine andcontains a 13 amino acid C-terminus tail was designed and synthesized.This polypeptide is referred to as an ASBNP.1 polypeptide (FIG. 1). Thebiological effects of intravenous ABNP.1 infusion were tested in normaldogs. Briefly, six normal dogs were infused with 2, 10, and 100 pmol ofan ASBNP.1 polypeptide preparation, i.e., each dog received consecutiveinfusions of 2, 10, and 100 pmol of an ASBNP.1 polypeptide preparation.Urine flow, urinary sodium excretion, distal fractional tubular sodiumreabsorption, proximal tubular fractional sodium reabsorption, meanarterial blood pressure, plasma cGMP levels, glomerular filtration rate,renal blood flow, and plasma renin levels were measured as describedelsewhere (Chen et al., Am. J. Physiol. Regul. Integr. Comp. Physiol.,288: R1093-R1097 (2005) and Haber et al., J. Clin. Endocrinol. Metab.,29:1349-1355 (2005)). Systemic administration of the ASBNP.1 polypeptideresulted in diuretic and natriuretic effects (FIGS. 2 and 3). Theeffects of the ASBNP.1 polypeptide targeted distal tubules (FIGS. 4 and5). Plasma cGMP was elevated at the super high dose. There was a trendtowards a decreased renin at the two higher doses (FIGS. 6 and 7).Systemic administration of the ASBNP.1 polypeptide had no effect onglomerular filtration rate, renal blood flow, or mean arterial bloodpressure (FIGS. 8-10).

These results demonstrate that the ASBNP.1 polypeptide has distinctrenal effects and lacks the ability to affect systemic blood pressure.

The following experiments were performed to compare the activities ofBNP, ASBNP, and ASBNP.1 polypeptides. Briefly, synthesized forms of BNP,ASBNP, and ASBNP.1 were administered to HUVECs and HASMCs, and cGMPlevels were determined. ASBNP had minimal effects, while ASBNP.1 had noeffect on cGMP in these cells. These results demonstrate that ASBNP andASBNP.1 had no effect when administered to a rabbit artery that waspre-constricted (FIG. 13) compared with BNP.

Example 2 Biological Effects of ASBNP.1 Polypeptides in Mammals with CHF

The biological effects of intravenous ABNP.1 infusion were tested in apaced dog model of congestive heart failure (CHF). Briefly, 10 dogsunderwent surgical implantation of a programmable cardiac pacemaker(Medtronic, Minneapolis, Minn.). After postoperative recovery, theanimals received 11 days of rapid ventricular pacing (240 beats/minute),which can induce overt congestive heart failure CHF as describedelsewhere (Chen et al., Circulation, 100:2443-2448 (1999)). The dogswere intravenously infused with 2, 10, and 100 pmol of an ASBNP.1polypeptide preparation, i.e., each dog received consecutive infusionsof 2, 10, and 100 pmol of an ASBNP.1 polypeptide preparation.

Acute hemodynamic studies were performed at the time of infusion, andcomparisons were made between groups and among dogs at baseline andduring each infusion. Urine flow, urinary sodium excretion, distalfractional tubular sodium reabsorption, proximal tubular fractionalsodium reabsorption, plasma cGMP levels, rennin levels, glomerularfiltration rate, renal blood flow, mean arterial blood pressure, urinarycGMP excretion, urinary ANP excretion, urinary BNP excretion, plasma BNPlevels, plasma ANP levels, pulmonary capillary wedge pressure, cardiacoutput, systemic vascular resistance, angiotensin II levels, andaldosterone levels were measured as described elsewhere (Chen et al.,Am. J. Physiol. Regul. Integr. Comp. Physiol., 288: R1093-R1097 (2005)and Haber et al., J. Clin. Endocrinol. Metab., 29:1349-1355 (2005)).Systemic administration of the ASBNP.1 polypeptide to the paced dogsresulted in diuretic but not natriuretic effects (FIGS. 15 and 16).Renin rate increased throughout the washout period (FIG. 20). Systemicadministration of the ASBNP.1 polypeptide increased glomerularfiltration rate at the high level (FIG. 21). Systemic administration ofthe ASBNP.1 polypeptide increased urinary cGMP excretion at the high andsuper high levels (FIG. 24). The high level also increased urinary ANPexcretion (FIG. 25). The super high level also increased urinary BNPexcretion (FIG. 26). Cardiac output was decreased from theadministration of the high level of polypeptide and remained decreasethroughout the remainder of the experiment (FIG. 30). Systemic vascularresistance was increased during the high, super high, washout, andrecovery 1 phases (FIG. 31). Aldosterone was increased from theadministration of the low level of polypeptide and continued to increasethroughout the remainder of the experiment (FIG. 33). There was noeffect on distal tubular fractional sodium reabsorption, proximaltubular fractional sodium reabsorption renal blood flow, plasma cGMP,mean arterial blood pressure, plasma BNP, plasma ANP, pulmonarycapillary wedge pressure, or angiotensin II levels (FIGS. 17, 18, 19,22, 23, 28, 29, and 32).

These results demonstrate that the ASBNP.1 polypeptide has renal effects(including enhanced GFR) and lacks the ability to affect systemic bloodpressure in CHF animals.

In another experiment, an ASBNP.1 polypeptide preparation wasadministered to dogs (100 pmol/kg/minute for 90 minutes). Urinary sodiumexcretion, urine flow, mean arterial blood pressure, renal blood flow,pulmonary capillary wedge pressure, and cardiac output were measuredafter 30, 60, and 90 minutes of administering the ASBNP.1 polypeptide.Administration of ASBNP.1 polypeptide was followed by a washout periodof 30 minutes. The washout was performed by administering normal saline.Urinary sodium excretion, urine flow, mean arterial blood pressure,renal blood flow, pulmonary capillary wedge pressure, and cardiac outputwere measured again after the washout period, and after each of tworecovery periods at 60 minutes (Rec 1) and 90 minutes (Rec 2) afteradministration of the ASBNP.1 polypeptide. The results are presented inFIGS. 34-39. Administration of ASBNP.1 polypeptide at a dose of 100pmol/kg/minute for 90 minutes was observed to increase urinary sodiumexcretion as well as urine flow (FIGS. 34 and 35). No significant effectwas observed on mean arterial blood pressure, renal blood flow, orcardiac output (FIGS. 36, 37, and 39). A decrease in pulmonary capillarywedge pressure was observed 60 minutes after administration of ASBNP.1polypeptide (FIG. 38), without a change in PAP (pulmonary arterialpressure) or RAP (right arterial pressure).

Example 3 Biological Effects of ASBNP.1 Polypeptides Using Animal Models

The effects of ASBNP.1 infusion is further assessed in the TIVCC model(a dog model of sodium retention which mimics cirrhosis and nephrosis).The TIVCC model of sodium-retention and ascites without concurrentincreases in cardiac filling pressure as described elsewhere (Wei etal., Am. J. Physiol., 273:R838-844 (1997)). The ASBNP.1 polypeptide istested in the TIVCC model using increasing doses up to 100pmol.kg/minute administered intravenously.

Example 4 Induction Radiocontrast-Induced Nephropathy in Dogs with HeartFailure Produced by Rapid Ventricular Pacing

Under pentobarbital anesthesia (30 mg/kg) and via a left thoracotomy andpericardiectomy, the heart is exposed and a screw-in epicardialpacemaker lead is implanted into the right ventricle. The pacemaker leadis connected to a pacemaker implanted subcutaneously in the chest. Inaddition, at the time of pacemaker implantation, a polyethylene catheter(PE 240, Clay Adams, Parsippany, N.J., USA) is placed via a femoralartery into the aorta at least 6 cm above the renal arteries. Dogs areallowed to recover over a three day period, during which time theyreceive prophylactic antibiotic treatment with clindamycin and Combater.Following recovery from surgery, the pacemaker is programmed to 250beats per minute and pacing continues at this rate for 10 days toproduce heart failure.

On the day of an acute experiment 11 days after starting pacing,radiocontrast agent (Vascoray®, Mallinkrodt, Inc., St. Louis, Mo., USA)is infused intravenously at a dose of 7 mL/kg over a 10-minute period.Dogs are returned to metabolic cages for a series of six consecutive24-hour urine collections for monitoring of radiocontrast inducednephropathy as described elsewhere (Margulies et al., KidneyInternational, 38(6):1101-8 (1990)).

In some experiments, ASBNP.1 polypeptide is administered to paced dogsprior to administering radiocontrast agent. Each dog is administeredconsecutive infusions of 2, 10, and 100 pmol of ASBNP.1 polypeptide orone 90 minute infusion with 100 pmol of ASBNP.1 polypeptide. Urinarysodium excretion, urine flow, mean arterial blood pressure, renal bloodflow, pulmonary capillary wedge pressure, and cardiac output weremeasured 30, 60, and 90 minutes after administering the ASBNP.1polypeptide, after a washout period, and during each of two recoveryperiods following the washout period. Measurements of the clinicalparameters are compared to measurements taken in control dogs that werenot administered ASBNP.1 prior to administration of radiocontrast agent.

Example 5 Prevention of Contrast Induced Renal Failure in High RiskPatients Including Patients with CHF

Patients at high risk of CIN (e.g., elderly patients and patients atrisk for chronic renal insufficiency, diabetes, and heart failure) whorequire contrast for imaging (e.g., angiography or CT) are treatedprophylactically with IV infusions of ASBNP polypeptide or ASBNP.1polypeptide prior to administration of contrast media. Prior to theinfusion, vital signs are taken and laboratory tests are performed tomeasure electrolytes, serum creatinine, cystatin, and BNP polypeptidelevels. BNP polypeptide levels are obtained using the Biosite BNP assay,which detects ASBNP polypeptide, and/or using an assay specific forASBNP polypeptide. Baseline urine output is measured and urineelectrolytes are assessed. An intravenous infusion of ASBNP polypeptide,ASBNP.1 polypeptide, or a derivative is initiated prior to contrastadministration. Vital signs and urine output are assessed every 2 hoursduring the infusion. At a predetermined time following initiation, thepatient undergoes contrast administration. This time may coincide withachievement of a polypeptide level of a certain concentration asdetermined by a specific assay for the polypeptides. Following contrastadministration, the infusion continues for 8 to 24 hours or until serumcreatinine and/or cystatin is noted to be unchanged from baseline.

Example 6 Treatment of Cardiorenal Syndrome

Patients who develop worsening renal function with diuretic resistancein the setting of acute decompensated heart failure are treatedprospectively with IV infusions of ASBNP polypeptide or ASBNP.1polypeptide. Prior to the infusion, vital signs are taken and laboratorytests are performed to measure electrolytes, serum creatinine, cystatin,and BNP polypeptide levels. Baseline urine output is measured and urineelectrolytes are assessed. An intravenous infusion of ASBNP polypeptide,ASBNP.1 polypeptide, or a derivative thereof is initiated. Vital signsand urine output are assessed every 2 hours during the infusion, whichis 12 to 72 hours in duration. Drug levels, BNP polypeptide levels,serum creatinine, cystatin, and plasma and urine electrolytes areassessed daily throughout the infusion.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a mammal having a renaldysfunction, wherein said method comprises administering to said mammala polypeptide between 37 and 47 amino acid residues in length, whereinsaid polypeptide comprises an amino acid sequence (a) set forth in SEQID NO:1 or (b) that aligns to the sequence set forth in SEQ ID NO:1 withfive or less amino acid additions, deletions, substitutions, orcombinations thereof, and wherein said administering is under conditionswherein the severity of a symptom of said renal dysfunction is reduced.2. The method of claim 1, wherein said mammal is a human.
 3. The methodof claim 1, wherein said renal dysfunction comprises renal failure. 4.The method of claim 1, wherein said renal dysfunction comprises renalfailure accompanied with congestive heart failure.
 5. The method ofclaim 1, wherein said polypeptide is between 37 and 47 amino acidresidues in length and comprises an amino acid sequence set forth in SEQID NO:1.
 6. The method of claim 1, wherein said polypeptide is between37 and 47 amino acid residues in length and comprises an amino acidsequence that aligns to the sequence set forth in SEQ ID NO:1 with fiveor less amino acid additions, deletions, substitutions, or combinationsthereof.
 7. The method of claim 1, wherein said symptom comprises anabnormal serum creatinine level, urine flow, renin level, glomerularfiltration rate, urinary cGMP excretion rate, urinary ANP excretionrate, urinary BNP excretion rate, cardiac output, systemic vascularresistance, or aldosterone level.
 8. A method for treating a mammalhaving a heart dysfunction, wherein said method comprises administeringto said mammal a polypeptide between 37 and 47 amino acid residues inlength, wherein said polypeptide comprises an amino acid sequence (a)set forth in SEQ ID NO:1 or (b) that aligns to the sequence set forth inSEQ ID NO:1 with five or less amino acid additions, deletions,substitutions, or combinations thereof, and wherein said administeringis under conditions wherein the severity of a symptom of said heartdysfunction is reduced.
 9. The method of claim 8, wherein said mammal isa human.
 10. The method of claim 8, wherein said heart dysfunctioncomprises heart failure.
 11. The method of claim 8, wherein said heartdysfunction comprises congestive heart failure accompanied with renalfailure.
 12. The method of claim 8, wherein said polypeptide is between37 and 47 amino acid residues in length and comprises an amino acidsequence set forth in SEQ ID NO:1.
 13. The method of claim 8, whereinsaid polypeptide is between 37 and 47 amino acid residues in length andcomprises an amino acid sequence that aligns to the sequence set forthin SEQ ID NO:1 with five or less amino acid additions, deletions,substitutions, or combinations thereof.